Polysaccharides (PS) represent major protective antigens on the surface of many pathogenic bacteria cells and have therefore been targets of successful and effective vaccines to prevent infectious disease. Conjugation of PS to proteins is currently the most established way to make immunogenic PS vaccines. However, conjugation technology is expensive and technically challenging. The Mekalanos laboratory has developed a simple and inexpensive way of making 'virtual conjugate vaccines' that employs Protein Capsular Matrix Vaccine (PCMV) technology. This proposal seeks to extend PCMV technology to modify killed whole cell bacteria vaccines (wcPCMV) and thus render them immunogenic after oral immunization and capable of inducing 'conjugate-like' immune responses against surface PS of bacterial cells. This will be accomplished by using a bifunctional molecular cross-linker (glutaraldehyde) to drive extensive proteinprotein coupling in the vicinity of surface-localized PS chains, thus entrapping the PS in a protein mesh that will remain associated with them after dissolution of the cell by elements of the host immune system. Thus, B cells expressing antibody receptors that recognize PS will take up PS only in the context of PCMV carrier protein. This process should (as with conjugate vaccines) trigger activation of T helper cells that instruct B cells to undergo replication, immunoglobulin class switching, and mutational affinity maturation of their anti- PS antibody genes. We will make wcPCMV vaccines for several species of organisms to control for the effect ofthe chemistry of different PS surface structures, and their density on the cell surface. These will include the O1 Inaba, O1 Ogawa and 0139 serogroups of Vibrio cholerae and Vi encapsulated Salmonella typhi. As a control for the properties of wcPCMV versus conventional PCMV and conjugates, we will also evaluate the method on several encapsulated types of Streptococcus pneumoniae, a Gram-positive bacterium. The wcPCMV vaccines will be explored as immunogens using both parenteral and mucosal routes of administration. The functionality of wcPCMV-induced antibodies will be assessed for complement dependent bacteriolysis activity, as well as their ability to protect experimental animals from direct pathogenic bacterial challenge. This proposal may lead to a multivalent wcPCMV vaccine against cholera, . brucellosis, 0157 E. coli, and anthrax. However, wcPCMV technology could more broadly impact mucosal vaccine development for many other mucosal bacterial pathogens that display PS on their surface.